Circulating microRNAs as potential biomarkers of physical activity in geriatric patients with HCV

Background Circulating microRNAs have been implicated in a diverse array of biological and pathological phenomena. Their potential utility as noninvasive biomarkers for screening and diagnosing various diseases has been proposed. Objective This study aimed to explore the potential role of the miRNAs miR-122 and miR-486 as molecular biomarkers in the pathogenesis of hepatitis C virus (HCV) infection. Thus, miR-122 and miR-486 were detected in the serum of HCV patients and healthy controls. Moreover, the potential correlations of miR-122 and miR-486 with viral complications, such as physical activity, pain, muscle fatigue, and HCV infection, were identified. Methods A total of 150 subjects aged 30 to 66 years were included in this study. The patients were classified as patients with chronic hepatitis C virus (CHC) (n = 110) or healthy controls (n = 40). Real-time polymerase chain reaction (PCR) analyses were performed to determine miR-122 and miR-486 expression. Physical activity (PA), pain score, HCV genotyping, viral overload, aspartate transaminase (AST), alanine transaminase (ALT), lactic acid dehydrogenase (LDH), creatine kinase (CK), and antioxidant status were also estimated by using prevalidated questionnaires, PCR, and spectrophotometric analyses. Results Compared with those in normal controls, significant increases in the serum levels of miR-122 and miR-486 were reported in patients with CHC. In physically active CHC patients, there was a significant correlation between the expression of miRNAs and increased alanine transaminase (ALT), aspartate transaminase (AST), fibrosis scores, and inflammation activity, but no association was reported for hepatitis C virus (HCV) RNA or viral load. Additionally, significant decreases in LDH, CK, GSSG, and pain scores and increases in TAC, GSH, and the GSH/GSSG ratio were reported. Moreover, the expression of miR-122 and miR-486 was positively correlated with changes in body mass index (BMI) and liver fibrosis stage, as well as negatively correlated with sex, PA, TAC, GSH, GSSG, and the GSH/GSSG ratio. Conclusion MiR-122 and miR-486 expression levels were strongly correlated with physical activity, pain perception, and muscle fatigue biomarkers in HCV-infected patients. These miRNA levels were associated with elevated AST, ALT, fibrosis scores, LDH, CK, and antioxidant status, thus suggesting their potential as biomarkers for disease severity and oxidative stress. However, no correlation was observed with viral load or HCV-RNA expression, thus implying that these miRNAs may impact disease progression and symptoms through host factors, rather than directly affecting viral replication. In summary, the results demonstrated that molecular studies of miR-22 and miR-468 and their associations with PA, pain, adiposity, sex differences, and muscle fatigue, as well as routine biomarkers, could be useful as prognostic nanoninvasive biomarkers, thus providing novel therapeutic targets for CHC infection.


Introduction
Chronic liver conditions, including liver cirrhosis, hepatic failure, and hepatocellular carcinoma, have been linked to chronic hepatitis C virus (HCV) infections in an estimated 170 million individuals worldwide [1,2].The progression of infection subsequently leads to severe liver complications, which can necessitate transplantation in many patients [3].Thus, eradication of HCV infection significantly reduces liver-related morbidity and mortality [4][5][6].
HCV infection has its most pronounced impact on patients' social and physical functioning, overall wellbeing, and vitality [7].Current medical advice recommends the management and correction of health-related behaviors, including nutritional or physical activity, during viral infection to prevent further liver disease progression.Importantly, this refers to the influence of lifestyle on disease development [8][9][10].
Patients who are afflicted with HCV infection have reported experiencing diminished self-assurance and a decrease in their physical capabilities [11,12].This decrease in quality of life, which is linked to chronic hepatitis, may be influenced by both comorbid conditions and the patient's awareness of their diagnosis rather than being solely dependent on the severity of the viral infection, liver function, or the extent of fibrosis [7,13].
The advantages of physical exercise are multifaceted and extend to mitigating risks in individuals with various chronic conditions [14][15][16][17].Indeed, physical exercise can play a crucial role in the management of chronic diseases [18].Earlier studies have suggested the incorporation of physical activity as an integral component in supporting individuals who are experiencing chronic HCV infection, with an aim of bolstering their self-assurance and overall capabilities [19,20].In physically active HCV patients, their bodies manufacture ATP more often, thus indirectly boosting the body's glutathione levels.
This scenario correspondingly improves the resilience of the liver against free radicals and hepatitis C damage [21].Using the aforementioned data, we can determine that there is no direct correlation between viral infection, disease progression, or physiological changes; moreover, only a correlation with antioxidant activity has been reported in patients with HCV [7,13,18,21].
Thus, in our hypothesis, we suggest that there is a relationship between physical activity and HCV infection, which may be clarified by investigating other biomarkers, such as microRNAs (miRNAs).MicroRNAs, which are short noncoding RNAs consisting of approximately 22 nucleotides, are involved in posttranscriptional gene expression regulation.They have been demonstrated to participate in a broad spectrum of biological and pathological processes [22][23][24].
Recently, there has been growing interest in assessing the potential of miRNAs as being noninvasive biomarkers for the screening and diagnosis of various diseases.The ability of circulating miRNAs to serve this purpose may be attributed to their presence in exosomes, which provide stability [25][26][27][28].In the human genome, over 2,500 miRNAs have been identified, and they are likely involved in a wide range of cellular processes, including (but not limited to) development, differentiation, proliferation, apoptosis, disease pathology, and antiviral defense [29,30].
In the context of HCV infection, several miRNAs are significantly upregulated in different HCV genotypes, with notable examples being serum miR-134, miR-198, miR-320c, and miR-483-5p, which show promise as being diagnostic biomarkers for HCV infection [31].Among all human miRNAs, microRNA 122 (miR-122) is one of the most highly expressed and abundant miRNAs, constituting more than 70% of the total miRNA content in the human liver.Consequently, it plays a pivotal role in the regulation of hepatic functions [32].Moreover, research has indicated that miR-122 is indispensable for HCV replication and may serve as a target for anti-HCV therapy [33].Additionally, there is a body of evidence linking physical activity to skeletal muscle development [34][35][36], and miRNAs are believed to act as physiological mediators of the adaptations induced by exercise [37].Thus, miRNAs represent interesting factors in the study of host-HCV interactions, ranging from HCV infection and related consequences such as low physical activity, fatigue, and depression to potential novel targets for antiviral therapy [38,39].
Additionally, several studies have shown that the molecular signatures of cellular miRNA expression may significantly control the classification, diagnosis, and prognosis of cancer [40,41].However, the diagnostic and prognostic accuracy of the miRNA genetic fingerprint for more than 16,000 mRNAs were significantly different in different cancers, including the incidence rates of oxidative stress.However, no correlation was observed with viral load or HCV-RNA expression, thus implying that these miRNAs may impact disease progression and symptoms through host factors, rather than directly affecting viral replication.In summary, the results demonstrated that molecular studies of miR-22 and miR-468 and their associations with PA, pain, adiposity, sex differences, and muscle fatigue, as well as routine biomarkers, could be useful as prognostic nanoninvasive biomarkers, thus providing novel therapeutic targets for CHC infection.
hepatocellular carcinoma among patients at high risk of hepatitis C virus infection [41,42].The biological functions and targets of several miRNAs, such as miR-618 and miR-650, have been reported as being noninvasive diagnostic markers [42] that can be identified in urine samples and are valuable approaches for developing screening methods that can reduce mortality rates among HCV-infected patients [43][44][45][46][47].
In addition, several studies have shown that cellular miR-486 is a multifunctional miRNA involved in many cancers, including the tumorigenesis of hepatocellular carcinoma, with additional functional roles as a modulator for regulating drug resistance, such as sorafenib resistance, by targeting FGFR4 and EGFR, thus offering a potential target for HCC treatment [44][45][46][47].Although the potential roles of both miR-22 and miR-486 as prognostic markers in the diagnosis, treatment, and prevention of drug resistance have been addressed in many studies [40][41][42][43][44][45][46][47], little is known about their association with physical activity, pain, and muscle fatigue as complications in patients with HCV infection.Thus, this study aimed to elucidate the potential role of miR-122 and miR-486 in relation to physical activity, pain, and muscle fatigue biomarkers in patients with HCV infection or its complications, as well as to provide valuable insights into the differences between patients with HCV infection and healthy individuals.

Study design
This study employed a case-control observational design to determine the relationships between miRNA-122 and miR-486 expression and physical activity, pain, and muscle fatigue biomarker levels in patients with HCV infection or its complications, as well as to compare these findings with those observed in the healthy control group.

Ethical considerations
The study protocol underwent review and received approval from the ethics subcommittee at King Saud University, Riyadh, Saudi Arabia, with the assigned file number ID: RRC-2016-045.The research adhered to ethical principles outlined by the local institutional review board and was conducted in accordance with the Declaration of Helsinki (2010).Prior to the commencement of the study, all of the participants were provided with comprehensive information about the research, and each participant provided their informed consent through a written and signed document.

Subjects
A total of 150 older adults, ranging in age from 30 to 66 years, met the eligibility criteria for participation in this study.The study included a control group consisting of 40 healthy HCV-negative individuals who exhibited normal levels of physical activity (with a PA score ≥ 2,500 MET minutes/week).Additionally, a total of 110 patients with chronic HCV infection, as well as with a confirmed disease duration of more than 10 years, viremia, HCV antibodies as indicated by the RIBA-II test, elevated serum transaminase levels surpassing the upper normal limit, HCV-RNA positivity, and genotype determinations, were included in the current study.The exclusion criteria included individuals with viral coinfections (HIV and/or HBV), advanced stages of other liver diseases (such as cirrhosis), hepatocellular carcinoma, prior liver transplantation, obesity (BMI ≥ 25 and ≥ 30 kg/m 2 ), iron overload, or insufficient liver biopsy results.Heparinized syringes were utilized to collect blood samples from all of the subjects either on the day of the biopsy or within 5 days following the biopsy procedure.Subsequently, serum samples were extracted through centrifugation, which lasted for 1 min at 1,400 rpm.These samples were assigned coded study identification numbers and were then promptly frozen at -20 °C for subsequent analysis.A comprehensive summary of the participants' demographic and clinical information can be found in Table 1.

Laboratory assessment
The levels of liver enzymes, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), and total bilirubin (TB), were measured in the sera of both healthy controls and HCV-infected patients.In addition, complete blood platelet count, glucose, and HOMA-IR were also estimated according to the manufacturer's instructions.Serum AFP levels were measured via sandwich enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN, USA).

Assessment of HCV infection
As a measure of diagnosis, HCV-RNA was also estimated qualitatively by using a nested PCR Qiagen RNA-extraction kit (Thermo Fisher Scientific, Waltham, MA, USA) and quantitatively by using Smart Cycler II real-time PCR (Cepheid, Sunnyvale, CA, USA) with HCV RNA quantification kits (Sacace Biotechnologies, Como, Italy) for estimation of HCV RNA-positive subjects, as previously described [48,49].Moreover, subjects with an HCV viral load lower than 250 IU/ml were considered negative.In addition, reverse hybridization was achieved to estimate the HCV genotype by using a line-probe assay (Inno-LiPA HCV II kit; Innogenetics, Ghent, Belgium) [50].
In this regard, patients with alanine aminotransferase (ALT) levels higher than the upper limit of normal, elevated titers of anti-HCV antibodies estimated with a third-generation enzyme immunoassay (AxSym HCV 3.0; Abbott Laboratories, Abbott Park, IL, USA), and confirmed levels of HCV-RNA by PCR analysis were diagnosed with chronic hepatitis C (CHC).

Histologic examination
The degree of liver inflammation in CHC patients was assessed through the use of the Batts and Ludwig scoring system [51].After informed consent documents had been submitted, hepatic biopsies were obtained from all of the patients by a surgeon after computed tomography or magnetic resonance imaging.The histological diagnosis of cirrhosis and HCC was based on an elevated serum AFP level (≥ 400 ng/mL) and computed tomography or magnetic resonance imaging scans, as recommended by international criteria.Thus, a preoperative clinical diagnosis of primary liver cancer and hepatocellular carcinoma (HCC) was made as described previously [52,53].
For histological examination, liver biopsies were obtained by using an automatic 16-gauge Trucut needle (biopsy gun), which provides adequate specimens for evaluation and fewer cases with tissue fragmentation.The analyzed liver biopsy specimens were at least 15-25 mm long with complete portal tracts (> 10).Formalin-fixed, paraffin-embedded sections were stained with hematoxylin and eosin and Masson's trichrome.Slides were labeled with patient identification numbers and subsequently reviewed and graded blindly by a senior pathologist.The mean length of liver biopsies and the number of portal tracts were assessed (including only the complete, intact portal tracts) [54][55][56].
The degree of fibrosis was scored according to the Metavir system, and no fibrosis was defined as F0, mild fibrosis as F1, moderate fibrosis as F2, severe fibrosis as F3, and cirrhosis as F4.Significant fibrosis was also defined as F2-F4.Hepatic inflammatory activity was also scored [54,55].

Isolation of miRNAs and RT-PCR
Total RNA from the serum samples of each subject was extracted by using TRIzol LS reagent (Invitrogen, Carlsbad, CA) and subsequently subjected to reverse transcriptase PCR (RT-PCR) analysis.A ready-made solution containing primers and probes for human miR-122 and miR-486 (Applied Biosystems, Foster City, CA) and real-time RT-PCR was generated by using an ABI 7300 system (Applied Biosystems) [46].After the addition of phenol solution to inhibit RNase activity, chemically synthesized cel-miR-39 (Sigma-Aldrich Japan, Tokyo, Japan) was added as an exogenous and endogenous control, and miR-16, which is a representative miRNA enriched in blood, was added as an endogenous control.For each serum sample, the ratio of the miRNA signal to that of endogenous (miR-16) and exogenous (cel-miR-39) controls was calculated as previously reported [57].

Serum biochemical analyses
A standard commercially available enzymatic assay (Granutest 15, EMD Millipore, Billerica, MA, USA) was used to calculate the serum creatine kinase (CK) concentration.Lactate dehydrogenase (LDH) activity was estimated by using an ultraviolet method provided by Randox Laboratories Ltd. (Antrim, UK).Serum alanine transaminase (ALT), aspartate transaminase (AST), albumin, bilirubin, blood platelet count, and fasting blood glucose were estimated in serum and blood samples by using standard methods.The serum total antioxidant capacity (TAC) was calculated by using a colorimetric assay kit (K274-100; BioVision, Milpitas, CA, USA).The data were measured and calculated as previously reported [58,59].The serum samples were utilized for spectrophotometric assays of glutathione (GSH) and glutathione disulfide (GSSG) levels, which was conducted by employing an enzyme-linked immunosorbent assay (ELISA) reader (Tecan GENios, A-5082, Tecan, Salzburg, Austria) and a glutathione assay kit (Cayman Chemical Company, Ann Arbor, MI, USA).The concentrations of GSH and GSSG were determined at 405 nm based on the color development of the TNB (5-thio-2-nitrobenzoic acid) product resulting from the reaction between GSH and DTNB (5,5'-dithio-bis-2-nitrobenzoic acid) [60].

Assessment of physical activity (PA)
A prevalidated global PA questionnaire (GPAQ) was used to calculate the PA scores of all participants regarding the intensity of exercise performed in minutes or hours per day for each participant, as previously reported [61,62].In relation to the intensity and frequency of PA achieved per week, energy expenditure was measured in the form of the metabolic equivalent (MET) of all of the participants.Thus, according to energy expenditure, PA scores were classified into three scores: physically inactive (MET minutes/week of ≤ 500), moderate PA (MET minutes/ week of 500-2,500), and physically active (≥ 2,500 MET minutes/week) [61,62].Using the PA scores as a criterion, HCV patients were divided into two categories: those who were physically active (≥ 2,500 MET minutes/ week, n = 85) and those who were PA inactive (MET minutes/week of ≤ 500, n = 25).

Assessment of VAS score
A reliable standard 100 mm visual analog scale (VAS), with 0 mm indicating "no pain" and 100 mm indicating "most severe pain", was used to measure the pain intensity of all of the participants, as previously reported [63,64].

Statistical analyses
The Shapiro-Wilk test was used to assess the normality of the distribution of the data, and logarithmic transformation was applied for subsequent statistical analyses.To evaluate differences between subject groups, both Student's t test and ANOVA analyses were utilized, followed by Bonferroni's multiple comparison analysis.To compare microRNA levels among different groups, adjustments were made using univariate analysis through a general linear model.Multiple stepwise regression and Pearson's correlation analysis were employed to determine the relationships between microRNA levels, TAC, LDH, CK, GSH/GSSG, HCV, and metabolic parameters.

Results
A study involving 150 participants was conducted, with the majority being male (66.66%).The clinical and demographic characteristics of both the control group and the CHC patients are presented in Table 1.CHC patients exhibited significantly greater values (P = 0.001) in terms of BMI, AST, ALT, platelet count, and glucose status than did control subjects.
In this study, LDH, CK, TAC, GSH, GSSG, and the GSH/GSSG ratio were measured as predictors of muscle function capacity and physical activity in controls and patients with CHC (Table 2).In physically active CHC patients, LDH, CK, and GSSG were significantly lower, whereas TAC, GSH, and the GSH/GSSG ratio were significantly greater than those in CHC patients with lower PA scores (P = 0.001) or in the control group (P = 0.01).Additionally, pain was significantly greater in patients with CHC who had lower PA scores than in both controls (p = 0.01) and PA patients with CHC (P = 0.001).
To explore the expression levels of miRNAs and their potential associations with physical activity patterns in CHC patients, we examined whether miR-122 and miR-486 were detectable and modulated in the serum of CHC patients.The serum concentrations of miR-122 and miR-486 were notably greater in all of the CHC patients (n = 110) than in the HCs (n = 40) (miR-122: 11.5-fold vs. 2.3-fold, respectively; P = 0.0001; miR-486: 42.6-fold vs. 21.9-fold,respectively; P = 0.0001) (Fig. 1A-B).
The correlations of miR-122 and miR-486 with demographic, physical activity, muscle capacity, antioxidant, and viral parameters were further studied by using stepwise regression and Pearson's correlation analyses (Table 3).Both miR-122 and miR-486 were positively correlated with glucose levels (P = 0.01) and BMI (p = 0.001) and negatively correlated with sex (p = 0.002).Furthermore, we assessed the activities of ALT and AST in CHC patients and observed a significant increase compared to those in control subjects.Additionally, we found that the relative expression of both miRNAs (miR-122 and miR-486) exhibited a moderately positive correlation with the serum ALT and AST levels (miR-122: r = 0.98, P = 0.001 and r = 0.78, P = 0.01, respectively; miR-486: r = 0.89, P = 0.001 and r = 0.92, P = 0.01, respectively) (Table 3).However, no correlation was detected between the expression levels of these miRNAs and either HCV-RNA (r = 0.215; P = 0.21) or the viral load Regarding physical activity and muscle capacity, the expression of miRNAs in CHC patients was positively correlated with LDH and CK and negatively correlated with PA score, TAC, GSH, GSSG, and the GSH/GSSG ratio (Table 3).In addition, pain intensity was significantly correlated with the levels of both miR-122 (P = 0.001) and miR-486 (P = 0.001).
Based on histological grading, the patients were categorized into two groups: those with an early stage of fibrosis (F0-F1) and those with advanced fibrosis (F3-F4).We found that the serum levels of miR-122 and miR-486 were significantly greater (P = 0.001) in patients with advanced liver fibrosis (miR-122, 10.8-fold; miR-486, 8.6-fold) than in those with early liver fibrosis (miR-122, 6.1-fold; miR-486, 5.7-fold) (Fig. 2).Notably, the increase in miR-122 expression was predominantly observed in patients with F2-F4 disease, whereas the increase in miR-486 expression was consistent with that in patients with F0-F3 disease and further elevated in patients with F4 disease.In addition, lower expression of the two miRNAs was reported in the livers of physically active patients than in those of patients with lower PA or sedentary lifestyles at comparable fibrosis stages (Table 4).

Discussion
MicroRNAs (miRNAs) are negative gene regulators with more than 2,500 copies in the human genome [21][22][23][24][25][26][27][28] and have been shown to control many crucial biological processes, such as cellular proliferation, cellular differentiation and apoptosis.The location of miRNAs in exosomes provides sufficient stability, thus underscoring  their role in diverse cellular processes, including disease pathogenesis and antiviral defense [29,30].In addition, an understanding of the pathogenesis of human virusassociated complications is important for developing effective means of preventing and treating liver diseases [65,66].Currently, the identification of noninvasive biological biomarkers, such as miRNAs, that can be used to screen high-risk patients for diagnosis is urgently needed [67,68].Changes in miRNA expression profiles have been documented in liver diseases, particularly in the context of HCV infection, spanning both the early and advanced stages of liver fibrosis [67,68].However, there is limited or no available information regarding the detection of miRNAs and their potential association with physical activity and muscle capacity in individuals with CHC.
Thus, the aim of our biomarker discovery approach was to investigate the potential of using miRNA expression in HCV-infected patients and its correlation with HCV complications in terms of physical activity, pain, and muscle capacity for potential use as noninvasive biomarkers for the early diagnosis of high-risk HCV.
In this study, we initially quantified the serum concentrations of both miR-122 and miR-486 in control subjects and individuals with CHC.Notably, patients with CHC exhibited markedly elevated serum levels of miR-122 and miR-486 in comparison to those in healthy controls.Our findings demonstrated positive correlations between miR-122, miR-486, alanine transaminase (ALT), aspartate transaminase (AST), fibrosis stage, and inflammation activity.However, we did not observe any significant correlations with HCV-RNA levels or viral load.Intriguingly, patients with advanced liver fibrosis (F3-F4) displayed higher expression levels of these measured miRNAs than did those with early liver fibrosis (F0-F1).
The release of miRNAs into the serum can occur through passive mechanisms, such as liver cell apoptosis and necrosis, or active processes involving the secretion of exosomes and viral particles.Consequently, the quantification of specific miRNA levels in serum may serve as a significant indicator of ongoing activity within liver cells [83,84].Although miRNAs are typically found within exosomes or macrovesicles, they can also exist in the form of free ribonucleoprotein complexes or be transported via the HBV surface antigen (HBsAg) [84][85][86][87][88].
Recently, it was postulated that the presence of miR-NAs enriched in muscles in the circulation may be associated with the modulation of metabolic responses induced by physical exercise [89].Previous studies have demonstrated that the levels of certain miRNAs in skeletal muscle can be altered by physical activity and exercise programs in humans [90] and experimental models [91].Previous reports have also suggested that to enhance the self-confidence and overall muscle capacity of patients with CHC, improving the physical activity of these patients is a promising option [19,20].The bodies of physically active patients with HCV manufacture more ATP, thus indirectly boosting the body's glutathione levels, which correspondingly improves the resilience of the liver against free radicals and hepatitis C damage [21].
In the current study, we attempted to determine whether there was an association between the expression of miRNAs and both physical activity and muscle capacity in controls and patients with CHC.In patients with CHC with a lower physical activity score, the serum levels of miR-122 and miR-486 were significantly lower than those in control subjects and more physically active patients with CHC.Consistent with our results, miR-486 was demonstrated to be significantly reduced in  physically active subjects following focused and regular exercise [92].However, in those with a sedentary lifestyle or who exhibit lower physical performance, increased levels of miR-486 were reported in the circulation [93,94].
The decrease in the release of miR-486 from muscle tissue into the bloodstream of physically active individuals may be linked more to the utilization of lipids rather than glucose as the primary energy source during physical activity [92,93].This phenomenon could involve the uptake of miR-486 from the blood into muscle tissue.Furthermore, this concept has been supported by previous research that explored the connection between HCV infection and lipid metabolism in the host.Notably, HCV proteins and circulating infectious particles have been associated with and shown to interact with lipid droplets and structures resembling very low-density lipoproteins (VLDLs), which are commonly referred to as lipoviral particles (LVPs).These interactions play a role in facilitating virion assembly and production [93, In addition, miR-122 was shown to be closely correlated with obesity, as were the levels of lipids, glucose and insulin in patients with a lower physical activity score; this effect correspondingly demonstrated a significant correlation with viral infection.In active patients, lipid utilization and decreased insulin resistance require the uptake of miR-122 from the blood into liver tissues [95,96].A wide range of miRNAs were up-or downregulated based upon individual participant, exercise type and the intensity of physical training, thus raising the possibility of manipulating and understanding tissue development, function, and disease progression [97,98].Furthermore, it was previously reported that miR-122 is one of the miRNAs that is solely expressed in the muscle, liver and brain [99][100][101].
In the present study, the levels of LDH, CK, TAC, GSH, and GSSG, as well as the GSH/GSSG ratio, were estimated as potential markers of physical activity and muscle capacity in patients with CHC and control subjects.
In physically active patients with CHC, significantly lower levels of LDH, CK, and GSSG and elevated levels of TAC and GSH were observed, as was a higher GSH/ GSSG ratio and a reduction in pain intensity compared to patients with CHC who had lower PA scores (P = 0.001) and those in the control group (p = 0.01).In addition, miR-122 and miR-486 expression in patients with CHC was positively correlated with LDH and CK levels and negatively correlated with PA scores; TAC, GSH, and GSSG levels; and the GSH/GSSG ratio.In physically active patients with HCV, their bodies manufacture more ATP, thus indirectly boosting the body's glutathione levels.This correspondingly improves the resilience of the liver against free radicals and hepatitis C damage [21].Normally, in liver disease, the antioxidant status is significantly reduced as a result of viral infection or hepatotoxic agents [102,103].
Reports indicate that HCV infection alone or in combination with HBV or HIV is significantly correlated with a reduction in cellular antioxidant mechanisms and elevated oxidative stress, which causes liver cell damage and a decrease in the number of mitochondrial DNA copies [104].Hence, the cumulative impact of oxidative stress and the depletion of antioxidants may play a crucial role in the progression of liver diseases [105,106].Generally, in patients with HCV infection, an increase in GSSG levels and a reduction in TAC and GSH levels and the GSH/ GSSG ratio have been reported in response to increased free radical oxidative species [107].
Additionally, physical activity through exercise has demonstrated effectiveness in decreasing inflammation and markers of oxidative stress in individuals with impaired liver function and liver fibrosis, as reported in previous research [86].Other studies have reported of elevated LDH and CK levels in HCV patients [85][86][87], and the release of miR-122 and miR-486 was shown to be linked to a noticeable increase in the peak LDH and CK levels in the muscle tissues of infected patients [108,109]; this miRNA expression was delayed following exercise or physical activity [89][90][91].
Concerning pain, previous research has indicated a connection between HCV infection and musculoskeletal pain, as well as general pain [92,93].In our investigation, we noted a substantial decrease in pain severity among CHC patients who were physically active.Previous studies have shown that physical inactivity is an additional negative parameter affecting chronic pain conditions [94][95][96][97][98], and patients or healthy persons who are regularly physically active may use moderate exercise as a significant tool for the prevention of chronic disease [14,15].Moreover, our results suggested that the expression of both miR-122 and miR-468 was negatively correlated with sex among the examined CHC patients, which suggests that miRNA expression could be influenced by sex differences.Previous studies have shown that some miRNAs expressed in certain tissues are sex specific and are significantly more strongly associated with the X chromosome in females than with the Y chromosome [110][111][112][113].Gender differences may be due to inactivation of one of the two X chromosomes occurring in men to maintain balanced expression of the X chromosomes, whereas chromosome inactivation leads to biallelic expression of miRNAs [110][111][112][113].In addition, significant sex differences in miRNA expression have emerged in many patients with liver diseases, including liver cancer and viral hepatitis, and are closely correlated with sex hormones [114,115].In addition, our results provided evidence that the expression of miR-122 and miR-468 was significantly associated with an increase in BMI, which is an adiposity marker, in CHC patients with poor physical activity compared to that in patients who are physically active.Previous studies have shown that adipose-derived exosomal miRNAs can regulate gene expression in distant tissues such as the liver and could be considered a novel class of adipokines [116][117][118].The importance of circulating miRNAs as novel adipokines in HCV infection remains to be clarified.
Obviously, further studies are necessary to clarify the mechanisms of action and targets of the identified miR-NAs miR-122 and miR-486, which are justified noninvasive biomarkers of the pathogenesis of HCV, as well as to correlate their expression levels with poor physical activity, obesity, sex, pain, and muscle fatigue, which are potential prognostic factors for CHC, and which can respond to therapeutic parameters.

Conclusions
In conclusion, this study demonstrated significant associations between miRNA-122 and miRNA-486 expression levels and various aspects of health in patients with HCV infection and other related viral complications in terms of physical activity, pain perception, adiposity, and muscle fatigue biomarkers.In CHC patients, the expression of cellular miR-122 and miR-486 was shown to play a crucial role in influencing physical activity, pain perception, adiposity, and muscle fatigue biomarkers within this CHC population.Furthermore, elevated levels of miR-122 and miR-486 were associated with increased levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and creatine kinase (CK).Additionally, these miRNA expression levels were correlated with fibrosis scores and antioxidant status, thus suggesting their potential utility as biomarkers for disease severity and oxidative stress in patients with HCV infection or its complications.Interestingly, no significant correlation was found between miR-122/miR-486 expression and viral load or HCV RNA expression in the same patients, thus suggesting that these microR-NAs may be more closely associated with overall health status and complications of HCV infection than directly impacting viral replication or RNA expression.
Overall, our study provides valuable insights into the potential of miR-122 and miR-486 as noninvasive biomarkers for evaluating the pathogenesis of CHC infection in terms of their correlation with physical activity, pain perception, adiposity, sex, and muscle fatigue in individuals with HCV infection.Further research should explore the mechanistic pathways underlying these associations and their clinical implications for managing HCV-infected patients.

Fig. 1
Fig. 1 Elevation in serum miR-122 and miR-486 expression in both physically active and non-active chronic hepatitis C. Serum levels of miR-122 (A) and miR-486 (B) were compared between control subjects and CHC patients.The CT values of serum miRNA expression levels were transformed into absolute values using standard curves.The bars represent the levels corresponding to CT values ranging from 10 to 12 (2.9 × 104 − 0.75 × 104 copies/ml) for miR-122 and 40 to 45 (1.75 × 104 − 0.68 × 104 copies/ml) for miR-486, respectively

Table 2
Change in muscle and antioxidant biomarkers, and pain scores (VAS) of control and geriatric CHC patients based up on PA (Mean ± SD).

Table 3
Stepwise regression analyses and Pearson's correlation of miR-122 and miR-486 with indices of HCV infectivity and muscle functional capacity in CHC patients r, Pearson's correlation coefficient; β, standardised regression coefficient; '-', indicates that the variable was not included in the multiple stepwise regression modelling.Abbreviations: CK, creatine kinase; LDH, lactate dehydrogenase; MET, metabolic equivalent; PA, physical activity; SD, standard deviation; TAC, total antioxidant capacity; VAS, visual analog scale; GSH, reduced glutathione; GSSG, oxidized glutathione

Table 4
Correlation between serum levels of miR-122 and miR-486 and histological liver severity in CHC patients